Compositions, methods, kits and apparatus for determining the presence or absence of target molecules

ABSTRACT

The present invention is directed to methods, compositions, kits and apparatus to identify and detect the presence or absence of target analytes. The embodiments of the present invention have utility in medical diagnosis and analysis of various chemical compounds in specimens and samples, as well as the design of test kits and apparatus for implementing such methods.

This application is a continuation in part application of U.S. Ser. No.08/801,154, filed Feb. 18, 1997 now U.S. Pat. No. 6,001,570.

FIELD OF THE INVENTION

The present invention is directed to methods, compositions, kits andapparatus to identify and detect the presence or absence of targetanalytes. The embodiments of the present invention have utility inmedical diagnosis and analysis of various chemical compounds inspecimens and samples, as well as the design of test kits and apparatusfor implementing such methods.

BACKGROUND OF THE INVENTION

Molecular biology advances in the last decade gave great promise for theintroduction of new, sensitive technologies to identify various analytesin test specimens, including the ability to diagnose cancer, infectiousagents and inherited diseases. Clinical molecular diagnostics dependalmost exclusively on restriction enzyme analyses and nucleic acidhybridization (Southern and Northern blots) (Meselson and Yuan, 1968,Southern, 1975). Clinical tests based on molecular biology technologyare more specific than conventional immunoassay procedures and candiscriminate between genetic determinants of two closely relatedorganisms. With their high specificity, nucleic acid procedures are veryimportant tools of molecular pathology. However, nucleic acid procedureshave limitations, the most important of which are the procedures consumetime, they are labor intensive, and have low sensitivity (Nakamura1993).

In any sample, the number of protein molecules of one kind is usuallyseveral times higher than the corresponding mRNA, and several hundredtimes higher than the number of genes encoding them. Usingantigen-specific antibodies is a routine procedure in the modemdiagnostic industry, although antibody development and purificationusually require laborious work. The specificity of tests based onmonoclonal antibodies depends on the capacity of antibodies todifferentiate between antigens, and might approach the specificity oftests based on nucleic acid hybridization. The sensitivity of thesetests, however, is routinely significantly lower than tests based onnucleic acid hybridization, even though the number of protein targetmolecules in each cell is relatively higher than the nucleic acidmolecules corresponding to them. It is desirable to use proteins as thetargets in diagnostic tests because of their abundance.

Thus, a need exists for improved diagnostic and analytical methods todetect the presence or absence of target molecules. A need also existsto detect non-nucleic acid analytes with nucleic acid chemistry. And,there is a need to detect protein targets with nucleic acid chemistrycoupled to a amplification system.

SUMMARY OF INVENTION

The present invention features methods, compositions, kits, andapparatus for determining the presence or absence of a target molecule.

One embodiment of the present invention is a composition. Thecomposition comprises a first ribonucleic acid (RNA) molecule and asecond RNA molecule. The first RNA molecule is capable of binding to atarget molecule and has the following formula:

5′—A—B—C—3′.

As used above, A is a section of the RNA molecule having 10-100,000nucleotides, which section is capable of being received by an RNAreplicase and with another RNA sequence, F, being replicated. The letter“B” denotes a section of the RNA molecule having approximately 10 to50,000 nucleotides, which section is capable of binding to the targetmolecule. The letter “C” denotes a section of the RNA molecule havingapproximately 1 to 10,000 nucleotides which section is capable of beingligated to another RNA sequence, “D”. The second RNA molecule is capableof binding to a target molecule and has the following formula:

5′—D—E—F—3′.

As used above, D is a section of the RNA molecule having approximately 1to 10,000 nucleotides, which section is capable of being ligated toanother RNA sequence, “C”. The letter “E” denotes a section of the RNAmolecule having approximately 10 to 50,000 nucleotides, which section iscapable of binding to the target molecule. The letter “F” denotes asection of the RNA molecule having 10-100,000 nucleotides which sectionis capable of being received by an RNA replicase and with anothersequence, “A”, being replicated. The first and the second RNA moleculesare capable of forming a third RNA molecule having the followingformula:

5′—A—B—C—D—E—F—3′.

The third RNA molecule is formed by ligation the C and D sections, asthe E and the B sections are bound to the target. The third RNA moleculeis capable of being received by an RNA replicase and being replicated bysuch enzyme.

Preferably, the sequences represented by the letters “A” and “F” areselected from the group of sequences consisting of MDV-I RNA, Q-beta RNAmicrovariant RNA, nanovariant RNA, midivariant RNA and modifications ofsuch sequences that maintain the ability of the sequences to bereplicated by RNA replicase. Preferably, the replicase is Q-betareplicase.

Preferably, the sections B and E each are sections having 10-5,000nucleotides and, even more preferred, 20-50 nucleotides. Preferably, thesections B and E bind to the target through non-nucleic acid basepairing interactions. And, preferably, the B and E sections are aptamersor partial aptamers as defined by Klug and Famulok (1994). Aptamers areselected for a particular functionality, such as binding to small orlarge organic molecules, peptides or proteins, the tertiary structure ofnucleic acids or complex or simple carbohydrates. The section B and Emay be derived from naturally occurring RNA exhibiting affinity forproteins. The sections B and E may also be engineered from computermodeling studies.

Preferably, the sections C and D each have 1-10,000 nucleotides, andmore preferred, 1-1000 nucleotides, and most preferred, 1-15nucleotides. Preferably, the sections C and D, when ligated together,define a recognition site for a ribozyme or a target of another compoundthat has an endonucleolytic activity against a single-stranded nucleicacid.

A further embodiment of the present invention features a method ofdetermining the presence or absence of a target molecule. The methodcomprises the steps of providing a first RNA molecule and a second RNAmolecule. The first RNA molecule is capable of binding to a targetmolecule and has the formula:

5′—A—B—C—3′.

The sections A, B and C are as previously described. The second RNAmolecule is capable of binding to the target molecule and has theformula:

5′—D—E—F—3′.

The sections D, E and F are as previously described. The method furthercomprises the step of imposing binding conditions on a samplepotentially containing target molecules in the presence of the first andsecond RNA molecules. In the presence of the target molecule, the firstand the second RNA molecules form a complex with the target molecule.The method further comprises the step of imposing RNA ligase reactionconditions on the sample to form a third RNA molecule in the presence ofthe target. The third RNA molecule has the formula:

5′—A—B—C—D—E—F—3′.

The sample is monitored for the presence of the third RNA molecule,presence or absence of which is indicative of the presence or absence ofthe target molecule.

Preferably, the sections B and E bind to the target through non-nucleicacid pairing interactions. And, most preferred, the B and E sections areaptamers or partial aptamers.

Preferably the sections C and D together comprise 5-15 nucleotides whichdefine a site for a ribozyme or a target of another compound that hasendonucleolytic activity against a single-stranded nucleic acid.

Preferably, at least one of the first or second RNA molecules has asignal generating moiety. After RNA ligase reaction conditions areimposed, the method preferably comprises the further step of separatingor enzymatically destroying the RNA molecules unbound with the targetmolecule and bearing the signal generating moiety.

Preferably, the signal-generating moiety is sections A and F of thethird RNA molecule, which sections allow recognition and replication byRNA replicase. Thus, the method further comprises the step of imposingRNA replicase conditions on the sample potentially comprising the thirdRNA molecule.

A further embodiment of the present invention comprises a kit fordetermining the presence or absence of a target molecule. The kitcomprises one or more reagents comprising a first RNA molecule, a secondRNA molecule and an RNA ligase. The first RNA molecule has the formula:

5′—A—B—C—3′.

The second RNA molecule has the formula:

5′—D—E—F—3′.

In the presence of the target, the first and the second RNA moleculesare capable of forming a target-first-and-second-RNA complex and in thepresence of RNA ligase means forming a third RNA molecule having theformula:

5′—A—B—C—D—E—F—3′.

The letters A, B, C, D, E, and F are as previously described. The thirdRNA molecule is preferably capable of being received and replicated byRNA replicase.

Preferably, the kit further comprises reverse transcriptase and suitableprimers.

An embodiment of the present invention further comprises a method ofmaking a first RNA molecule and a second RNA molecule, wherein the firstRNA molecule has the formula:

5′—A—B—C—3′

and the second RNA molecule has the formula:

5′—D—E—F—3′.

As used above, the letters A, B, C, D, E, and F are as previouslydescribed, and the sections C and D together define a site for cleavageby ribozyme or another compound having endonucleolytic activity againsta single-stranded nucleic acid.

The method comprises the step of combining a sample containing thetarget molecule with a library of RNA molecules having the formula:

5′—A—B′—C—D—E′—F—3′

to form a mixture of one or more target bound-RNA molecules and one ormore unbound-RNA molecules. The letters B′ and E′ represent potentialsections B and E.

Preferably, the sections B′ and E′ are randomized nucleotides.Optionally, as an alternative, primer nucleic acid corresponding to atleast one section is added to the mixture with an enzyme capable ofdegrading the unbound RNA molecules. However, this step may be omittedand the bound RNA molecules amplified as set forth is the next step.

Next, bound RNA molecules are released from the target and amplifiedwith RNA replicase, and preferably Q-beta replicase, to form anamplification product. This amplification product can be seriallydiluted and amplified, repeatedly if needed, to identify a preferredamplification product. Next, the RNA molecules comprising theamplification product having the formula:

5′—A—B′—C—D—E′—F—3′

are cleaved to form the first and second RNA molecules.

Preferably, the cleavage is performed with a ribozyme or otherendonucleolytic enzyme and the sections C and D together define acleavage site for the ribozyme or another endonucleolytic enzyme.

Preferably the step of degrading the unbound RNA molecules is performedin the presence of the enzyme reverse transcriptase. Preferably, thestep of amplifying the bound RNA molecules is performed in the presenceof the enzyme Q-beta replicase.

An embodiment of the present invention further comprises a kit forperforming the above method of identifying first and second RNAmolecules. The kit comprises one or more nucleic acid molecules havingsections corresponding to the sections A, B, C, D, E, and F. Preferably,the kit comprises sections B′ and E′ as randomized nucleotide sequences.

As used herein the term “kit” refers to an assembly of parts,compositions and reagents with suitable packaging materials andinstructions.

The present invention is further described in the following figure andexamples, which illustrate features and highlight preferred embodimentsand the best mode to make and use the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts plasmid pT7 MDV-XhoI. This plasmid was used for cloningsynthesized PX and XS dsDNAs and for the transcription of the PX and XSrecombinant RNA molecules that served as detector molecules foradenovirus sequences;

FIG. 2 depicts a hybridization product;

FIG. 3 depicts an aptamer bound to target; and

FIGS. 4a and 4 b depict an affinity product and a ligation product.

DETAILED DESCRIPTION OF INVENTION

The present invention will be described with respect to a SELEX processand Q-beta technology. SELEX processes are described more fully inseveral references (King and Famulok, 1994) (Ellington and Szostak,1990) and (Gold et al 1995). The method features the incorporation ofspecially-designed RNA libraries into a Q-beta replicase template. Themethods of the present invention are more effective than other methodsknown in the art for several reasons.

First, Q-beta replicase can only use templates with high structuralcomplexity, which are the best candidates for aptamers with greataffinity for the target molecule. The original library is pre-selectedfor the species with secondary and tertiary structures when the originallibrary of randomized sequences is amplified with Q-beta replicase priorto its contact with the target molecule.

Secondly, the special design of the libraries will allow amplificationof the high affinity ligands by enzymatic degradation of the lowaffinity molecules discriminately and selectively without an elaborateprocess of partitioning them from ligands of low affinity.

Thirdly, amplification of the ligands by Q-beta replicase as a part ofthe chimeric template RNA seems more manageable technically and moretime advantageous than using routine PCR techniques during standardSELEX procedures.

Finally, Q-beta chimeric RNA template with an insert of RNA ligandshighly specific for a target protein can serve directly as a unique toolto detect in clinical specimens target molecules having tertiaryconfigurations.

The embodiments of the invention have application for constructingspecially designed nucleic acid detector molecules for any analytes,including proteins, that naturally exhibit an affinity for nucleic acidmolecules and, especially, RNA molecules. The methods, compositions andkits have utility in the study of RNA-protein interactions and theirsignificance in regulatory systems, for diagnosis of cancer, infectiousand inherited diseases. The list of naturally-occurring andmedically-important RNA-protein complexes includes, but is not limitedto, RNA-binders of bacteria and parasites, intron binding proteins, RNAepitopes in autoimmune diseases, protein-nucleic acid complexes ofspinal muscular atrophy or in fragile-X mental retardation andRNA-protein subunits in telomerase. Naturally occurring nucleoproteincomplexes also include any regulatory proteins, enzymes, antibodies,antibiotics and other complex chemical compounds, as well as simplercomplexes, such as nucleotides, nucleosides or amino acids and such, forwhich naturally-occurring nucleic acid ligands have been identified anddescribed. The proposed method is also useful for any target moleculefor which nucleic acid ligands were obtained through SELEX, in vitroselection or in vitro evolution procedures.

The embodiments of the present invention feature a method forconstructing the sets of two detector molecules for identifying variousanalytes different from nucleic acid targets in a specimen. Thecomposition of the detector molecules is based on a ligand's nucleotidesequences, and the secondary and tertiary structure of the ligandcorresponding to such target analyte. The detector molecules mean twoRNA molecules that serve three purposes: to be a ligand with highaffinity to the target analyte, to be ligated into one functionalmolecule, and to be a template that could be amplified by Q-betareplicase after its ligation by RNA ligase. The specimen means anysample taken from any source and preferably, from biological sources.Target analyte means any compound of interest without limitation. Ligandmeans a nucleic acid molecule that demonstrates high affinity to thetarget analyte.

One embodiment of the present invention features a method for designingand constructing two detector molecules representing the nucleic acidcomponent of a nucleoprotein complex molecule or a ligand of knownnucleotide sequence. The detector molecules are constructed throughcloning them as appropriate oligonucleotides in the recombinant plasmidusing a Q-beta replicase template cDNA insert or synthesizing them on aDNA synthesizer, or using PCR techniques.

To construct the detector molecules for a target analyte with a knownligand, two sets of complementary oligonucleotides are designed andsynthesized on a DNA synthesizer. One set of oligonucleotides is dsDNArepresenting the 5′ part of the whole ligand. The other set ofoligonucleotides is dsDNA representing the 3′ part of the same ligand.Both dsDNAs are designed with terminal restriction enzyme sites forcloning in the vector, and with additional nucleotides with lengths fromone to ten nucleotides. These additional sequences will be used insubsequent steps of the procedure to synthesize captomers (from Lat.captere—seek to get), parts of the molecules with a specially-designedfunction. The first dsDNA has the following formula:

M——N——O——P.

The second dsDNA has the following formula:

P——R——S——T,

where M, P and T are restriction site linkers, O is sequencesrepresenting the 5′ segment of the ligand, R is sequences representingthe 3′ segment of the ligand, and N and S are the sequence with donorand acceptor termini employed in a ligation reaction.

These two dsDNAs are cloned in a recombinant plasmid containing T7 RNApromoter, followed immediately by inserting a Q-beta replicase templatecDNA (FIG. 1). Three unique restriction sites (M, P and T) for cloningdsDNA molecules are incorporated into the recombinant plasmid. Onecloning site, M follows the T7RNA promoter immediately. The T cloningsite is inserted into the end of the Q-beta replicase template, and theP site divides the template insert into two, 5′ and 3′, parts. Thus, the5′ part of the Q-beta replicase template is flanked by M and Prestriction sites and 3′ part of the template is flanked by P and Trestriction sites.

The composition of the insert in such recombinant plasmid will be:

T7 promoter—M—Q-beta template—P—Q-beta template—T

A second recombinant plasmid is prepared by replacing the 5′ part of theQ-beta replicase template cDNA situated between the M and P restrictionsites with corresponding dsDNA representing the 5′ segment of theligand. The combined insert of the second recombinant plasmid has thefollowing formula:

T7 promoter——M——N——O——P——Q-beta template——T.

A third recombinant plasmid is prepared by replacing the 3′ part of theQ-beta replicase template cDNA situated between the P and T restrictionsites with corresponding dsDNA representing the 3′ segment of theligand. The combined insert of the third recombinant plasmid has theformula:

T7 promoter—M—Q-beta template—P—R—S——T.

Recombinant plasmids containing the template sequences with the insertedsequences are used to transform competent bacterial cells, and thetransformed cells are grown in a culture. The cultured cells areharvested and lysed. The DNA plasmids are purified. In the said methodfor design and construction of the first and second detector-molecules,the recombinant plasmids are cleaved with an appropriate restrictionenzyme and the recombinant Q-beta replicase templates containing theinserts are transcribed into the RNA using T7 RNA promoter. Allprocedures are performed according to the standard protocols of Sambrooket al., (1989) known to someone skilled in the field of molecularbiology.

The second and third recombinant plasmids will be linearized by cleavagein the T restriction site, and the recombinant RNAs will be transcribedfrom each plasmid using the T7 RNA promoter.

Two recombinant RNA transcripts are the set of detector-molecules forthe chosen analyte.

Each such RNA detector-molecule consists of three segments. Thestructure of the first detector-molecule is:

5′—A—B—C—3′.

And the structure of the second detector-molecule is:

5′—D—E—F—3′.

Each component has one defined function-amplification, recognition orligation. The A and F “amplification” segments of the first and seconddetector-molecules are parts of a template replicable by Q-betareplicase. The nucleotide composition and the length of the A and Fsegments depends on the replicable RNA they represent. Preferably,segments A and F are selected from the group of replicable RNAsconsisting of MDV-I RNA, Q-beta RNA, microvariant RNA, midivariant RNA,nanovariant RNA, or modifications thereof that permit the RNA tomaintain its reproducibility. Neither the A nor the F parts of theQ-beta template can separately serve for amplification by Q-betareplicase.

A preferred sequence for section A is set forth in SEQ ID No. 1 below:

SEQ ID No. 1

5′-GGGGACCCCC CCGGAAGGGG GGGACGAGGU GCGGGCACCU CGUACGGGAG UUCGACCGUGACGCUCUAG-3′

A preferred sequence for section F is set forth in SEQ ID No. 2 below:

SEQ ID No. 2

5′-AGAUCUAGAG CACGGGCUAG CGCUUUCGCG CUCUCCCAGG UGACGCCUCG UGAAGAGGCGCGACCUUCGU GCGUUUCGGU GACGCACGAG AACCGCCACG CUGCUUCGCA GCGUGGCUCCUUCGCGCAGC CCGCUGCGCG AGGUGACCCC CCGAAGGGGG GUUCCC-3′

Sequences Nos. 1 and 2 are the sequences of MDV-1 RNA template forQ-beta replicase.

In the alternative, a preferred sequence for section A is set forth inSEQ ID No.3 below:

SEQ ID No. 3

5′-GGGGAAAUCC UGUUACCAGG AUAACGGGGU UUCCUCA-3′

And, a preferred sequence for section F is set forth in SQ ID No. 4below:

SEQ ID No. 4

5′-CCUCUCUACU CGAAAGUUAG AGAGGACACA CCCGGAUCUA GCCGGGUCAA CCCA-3′

The B and E “recognition” segments represent the full length or part oftwo ligands with high affinity to two epitopes of the same analyte, or,they are two parts of a single nucleic acid ligand with high affinity toa single epitope of the target analyte or nucleic acid component of thenucleoprotein complex. Segments B and E are the RNA transcripts of the Rand O parts of the ligand described above.

The D and C “ligation” segments termed captomers (from Lat. captere—seekto get) are segments of the detector molecules, with specially-designeddonor and acceptor terminal nucleotides that are essential for ligatingthe two detector molecules after they bind with the target molecules,the termini of which are used by RNA ligase in the ligation reaction toform phosphodiester bonds. The lengths of these segments can be as shortas one nucleotide and as long as 10,000 nucleotides. These two segmentsare the RNA transcripts of the captomer parts N and S of the recombinantdsDNA molecules. Neither of these two recombinant RNA molecules canseparately serve as a template for Q-beta replicase.

The synthesis of dsDNA, the cloning and the transcription of therecombinant RNA molecules are performed under the conditions describedin detail (Sambrook et al., 1989) and known to someone skilled in thefield of molecular biology.

Another embodiment of the present invention features a method fordesigning and constructing a pair of detector-molecules for anythree-dimensional analyte when the nucleotide sequence of its ligand isnot known. The method comprises the steps of providing a firstdetector-molecule and a second detector-molecule.

Preferably, the process starts from constructing machine-synthesizedlibraries of deoxyribooligonucleotides. Each member of each library iscomposed of at least the four segments:

Where the B and E segments are approximately 5′—B—CD—E—X—3′, 10-50,000nucleotides and, most preferably, 20-50 nucleotides each, and the CDsegment is of 1 to 10,000 nucleotides and, most preferably, 2-30sequences, and X is 10-15 nucleotides long.

Preferably, the B and E segments are random nucleotide sequences whereA, T, C and G nucleotides have an equal probability to be incorporatedin any position of the segment. Each member of the synthesized libraryis different from others in these two regions.

Preferably, the C—D segment contains DNA sequences that are arecognition site for a known ribozyme defined and described by O.Uhlenbeck (1987) as well as by G. F. Joyce (1989), T. R. Cech, F. L.Murphy, A. J. Zaug, C. Grosshans, U.S. Pat. No. 5,116,742 (1992), J. P.Hazeloff, W. L. Gerlach, P. A. Jennings, F. H. Cameron, U.S. Pat. No.5,254,678 (1993), H. D. Roberson and A. R. Goldberg U.S. Pat. No.5,225,337 (1993) or any other compound that demonstrates a specificendonucleolytic activity with single-stranded nucleic acid molecules.The segment with ribozyme recognition sequences is identical in allmolecules of the same library, but varies among the libraries.

Preferably the X segment is a DNA oligonucleotide segment with random,but defined sequences. Each molecule of the synthesized library has thesame X region.

The single-stranded original library will be converted into adouble-stranded DNA library using any DNA polymerase that includes, butis not limited to DNA polymerase I, Klenow fragment, T4 DNA polymerase,T7 DNA polymerase, and primers complementary to the X section of thesynthesized molecules. The conversion of the ssDNA library into a dsDNAlibrary, primer and enzymes is performed under standard conditions knownto someone skilled in the field of molecular biology.

The dsDNA library is cloned into a recombinant plasmid (similar to theone described previously, FIG. 1) containing the T7 RNA transcriptionpromoter attached to a cDNA copy of the Q-beta replicase template withan insert of a unique restriction enzyme linker within the sequence ofwhole recombinant plasmid, including the Q-beta replicase template.Insertion of the library sequences into the Q-beta template at theposition chosen must not unduly perturb the features of the templatenecessary for successful amplification. Insertion of the dsDNA libraryinto a recombinant plasmid containing a cDNA copy of Q-beta replicasetemplate is performed under standard conditions known to someone skilledin the field of molecular biology.

Recombinant plasmids containing the template sequences with the insertedsequences from the original DNA library are used to transform competentbacterial cells, and the transformed cells are grown in a culture. Thecultured cells are harvested and lysed. The DNA plasmids are purified.In the said method for design and construction of the first and seconddetector-molecules, the recombinant plasmids are cleaved with anappropriate restriction enzyme and the recombinant Q-beta replicasetemplates containing the inserts of the original DNA library aretranscribed into the RNA library using T7 RNA promoter. All proceduresare performed according to the standard protocols of Sambrook et al.,(1989) known to someone skilled in the field of molecular biology.

Composition of each species of the second RNA transcripts library, is5′—A—B—C—D—E—F—3′.

Each species is a molecule with three functional parts. The A and F aretwo parts of a single Q-beta template and enable amplification of thewhole detector molecule. The B and E segments represent random sequencesof the library in the recombinant RNA transcript. The CD segment of eachRNA transcript is a joining region of the B and E sequences and containsa recognition site for a chosen ribozyme or any other compound thatdemonstrates the cleavage of the single-stranded nucleic acid-specificsequences. The X segment is not essential for further procedures and itis not described in further descriptions.

A further embodiment of the present invention describes a method ofenrichment of the original RNA library with RNA species that demonstratesecondary and tertiary complexity in the B and E regions. Therecombinant transcript-RNAs' library templates are used to initiateamplification in a standard Q-beta replicase reaction according to thestandard procedure described in detail by Axelrod et al., (1991). Thesecondary structure of the RNA templates has a very powerful influenceon replication by the Q-beta enzyme. In fact, the existence andcontinual propagation of whole recombinant RNA species asself-replicating entities depends on these structures as the majorfactor in determining the viability of the amplification products.Q-beta replicase discriminates the inserts as a part of the templatemolecule on the basis of their secondary structures and provides apositive selection favoring those templates containing inserts with morecomplex secondary structures, as follows from Axelrod et al., (ibid.).Thus, after several, preferably three or four, cycles of replicatingwith Q-beta replicase, the library of recombinant RNA templates will be“preselected” for those recombinants with a higher proportion of thecomplex secondary structures that result from nucleotide pairing andinteraction.

Additionally, the “preselection” process for spatial complexity of B andE region will lead to an additional diversity of the original RNAlibrary. Since Q-beta replicase can use both, plus and minus strands ofRNA as templates, the “preselected” library will consist of two types ofmolecules that are plus and minus versions of the same sequence.

The secondary structure motifs contain such elements as pseudo knots,simple stems or hairpins, or stems with loops, or symmetrical andasymmetrical bulges and such. Double-stranded RNA of the B—CD—E partwith motifs of secondary structure in turn form a three dimensionalstructure, the tertiary structure of the recombinant RNA in this region,converting the B and E segments into segments with randomizedthree-dimensionally-structured nucleotide sequences. The relationshipbetween nucleotide sequences in RNA molecules, and secondary andtertiary organization of RNA molecules is described in detail by L. Goldand C. Tuerk, U.S. Pat. No. 5,475,096 (1995).

The further embodiment of the present invention features a procedure forselecting recombinant RNA species that demonstrate high affinity to atarget molecule of particular functionality, such as binding to small orlarge organic and non-organic molecules with their own tertiarystructures, including peptides or proteins, nucleic acid molecules oroligonucleotides, nucleotides or amino acids, complex or simplevitamins, antibiotics and carbohydrates and such without limitation. Theprocedure comprises the steps of contacting the library with the targetmolecules, providing an annealing of the detector-molecules preselectedfor tertiary structure with a population of the said target moleculesunder conditions favorable for forming the complex between the chosentarget and recombinant RNA molecules. The annealing will be performedunder favorable conditions described by Gold et al., (1995).Specifically, the recombinant RNA molecules will form a binding complexwith the target molecule in the 5′—B—CD—E—3′ region, and particularly,in the B and E segments. Preferably, sections B and E bind throughnon-nucleic acid base pairing interaction to the analyte. These twosegments may anneal to the same “epitope” of the target molecule, actingas two parts of a single nucleic acid ligand or, as an alternative, theB and E segments can act as two separate ligands forming bonds with twoseparate “epitopes” of the same target molecule. The CD region may ormay not participate in the annealing with the target molecule, dependingon its nucleotide constitution, nature of the target molecule and itsinteraction with the B and E segments..

Several possible configurations of molecules are in the mixture aftercompleting an annealing reaction: free RNA species, free targetmolecules and RNA/target molecule complexes. Additionally, RNA/targetmolecules complexes can be formed by annealing both B and E segments tothe analyte molecules, or either of them, B or E, annealed to theanalyte molecule separately. Several methods suggested for partitioningthe free RNA species from the target/RNA complexes, including filterbinding, gel mobility shift, affinity chromatography, antibodyprecipitation, phase partitioning and protection from nucleolyticcleavage by catalytic RNAs, termed ribozymes, have been described indetail by L. Gold and C Tuerk, U.S. Pat. No. 5,475,096 (1995). Further,L. Gold and S. Rinquist, U.S. Pat. No. 5,567,588 (1996) widened themethods that should be employed for partitioning nucleic acid moleculesannealed to the target analyte from those that do not bind the targetmolecules. Their new method, termed solution SELEX, employs a primerextension inhibition, exonuclease hydrolysis inhibition, linear tocircular formation, and single stranded nucleic acid PCR amplificationfor the partitioning between high- and low-affinity nucleic acid-targetcomplexes.

Preferably, the third RNA molecules are amplified by Q-beta replicase.The amplification product is serially diluted and subjected toadditional amplification to render the unbound, unamplified nucleicacids as mere background. This step of dilution and amplification can berepeated until the one or more desired nucleic acids are present in aconcentration much greater than any other components. However,additional selection steps may be applied as described below.

A further embodiment of the present invention is based on the enzymaticselection of the recombinant RNA template with high affinity to targetmolecules of the B—CD—E region embedded between two sectors, A and F, ofthe Q-beta template. To initiate this selection, we use a reversetranscriptase (RT), preferably Avian myeloblastoma virus (AMV) reversetranscriptase and an RT primer with nucleotide sequences complementaryto a segment of CD region. Reverse transcriptase from avianmyeloblastosis virus is a DNA polymerase that catalyzes polymerizationof nucleotides using an RNA template. This enzyme consists of twopolypeptide subunits, one of which contains 5′-3′ polymerase activityand the other a powerful RNase H activity (Verma, 1991). Reversetranscriptase has been widely used to synthesize complementary DNA(cDNA). Such synthesis requires a primer and free nucleotides. The RTenzyme will synthesize cDNA and simultaneously degrade the complementaryRNA molecule because of its RNase H activity. RNA with high affinity tothe target analyte molecule will be protected from degradation due tothe fact that the primers do not bind and RT cannot synthesize cDNA.Therefore RNase H has no activity.

The dissociation constant of the high affinity detector RNA-targetanalyte complex is in the nanomolar-or-less range and efficientlyinhibits annealing of the primer with complementary sequences of the Cand D regions bound to two separate epitopes or with one epitope, and,possibly, in configuration when only the C or D region is bound to asingle epitope. The success of the RT primer annealing with the RNAmolecule in the latter configuration will depend on the energy balancebetween the epitope-RNA and the primer-RNA complexes. At the same time,the RT primer will anneal efficiently with the complementary sequencesof the CD region of the free recombinant RNA molecule. As a result,reverse transcriptase synthesizes the cDNA strand using the freerecombinant RNA molecule only, and in those RNA-target complexes wherethe energy balance shifts in a direction favorable for creating aprimer-RNA complex. In the process of synthesizing cDNA, reversetranscriptase degrades the original RNA in RNA-DNA hybridsexonucleolytically because it has RNase H activity. Thus, a largesection of the recombinant RNA molecules with low affinity to thetarget, starting from the CD section to the end of the whole RNArecombinant molecule will be degraded. As a result, all low affinity andpartially annealing to the analyte RNA molecules will be eliminated fromthe ampliflable pool, and only completely annealing with target analyterecombinant RNA molecules with high affinity for the B—CD—E region willrepresent the pool amplifiable by a Q-beta replicase template.

Using enzymatic degradation of RNA molecules with low affinity to thetarget by RNase H allows isolation and consequent dissociation of thehigh affinity RNA-target molecules complex and purification of the highaffinity RNA molecule(s) from the target molecules. Purification of thewhole-length recombinant RNA molecules from the target is performed inconditions favorable for each target-RNA molecule combination. PurifiedRNA recombinant molecules with high affinity to the target are amplifiedby Q-beta replicase, using the standard protocol described further inthe EXAMPLE section.

A further embodiment of the present invention features the proceduresfor producing detector molecules from the product of the describedenzymatic degradation of low affinity RNA species and propagation ofrecombinant RNA molecules that demonstrate high affinity to the chosentarget analyte. All members of the remaining population will be exposedto an appropriate ribozyme or another agent that specifically cleavesthe CD sequence. Each RNA molecule will be split into twomolecule-detectors. The first molecule-detector will be composed of thesectors 5′—A——B——C—3′. The second molecule detector will have the orderof the segments comprising it as 5′—D——E——F—3′. The function of eachsegment was described earlier, except that each of the detectormolecules will have a new component-a terminal C or D region that waspreviously united in the CD region of the transcribed recombinant RNAmolecule. These two regions acquire a new important function in thedetector-molecules and will serve as the donor-acceptor captomers, theparts of the ligation complex.

The constructed set of two detector molecules is ready for use indetecting target analytes in clinical specimens.

In a modified variant, the detector molecules are ssDNA molecules withthe composition of the segments similar to those of the RNA nature. Bothdetector molecules contain the sequences representing the two parts ofthe Q-beta replicase template, the detection parts with high affinity tothe analyte molecule and the sequences recognizable by a DNAendonuclease. One of the detector molecules further includes an RNApolymerase promoter sequence to enable transcription of the DNAdetector-molecules annealed to the target and ligated into a recombinantRNA molecule. The organization of such recombinant RNA is the same aswas described previously, that is, restriction enzyme sequences areflanked by two analyte recognition sequences and those sequences areflanked by Q-beta replicase template sequences. This recombinant RNAmolecule is a template that will be amplified by Q-beta replicase.

A further embodiment of the present invention features the proceduresfor detection of an analyte molecule in a specimen using a constructedset of detector molecules. The constructed detector molecules could beused in diagnostic tests, in diagnostic test kits and for microsensorsor nucleic acid biochip production. For detection of an analytemolecule, a mixture of two detector-molecules is combined with aspecimen potentially containing the target analyte. The target, whenpresent in the specimen, will bind the detector-molecules, and the twohalves of the original recombinant RNA molecule will be situated on thetarget analyte in such a position that they can be ligated with RNAligase. After ligation, which occurs between the 3′ and 5′ terminalnucleotides of the C and D sectors, the two detector molecules form asingle molecule. Such molecules that combine the A and F sectors of thedetector molecules, can serve as a template for Q-beta replicase.Template ability is restored after ligation of the detector moleculesinto a single reporting recombinant template molecule that can then beamplified by Q-beta replicase.

Preferably, the two RNA detector- probes are joined or ligated whenbound to the target by an enzyme. One such enzyme be RNA ligase, andpreferably T4 RNA ligase, originally described by Leis et al., (1972).Similarly to DNA ligase, T4 RNA ligase catalyzes the formation of a3′♯5′ phosphodiester bond between a 3′-terminal hydroxyl and a5′-terminal phosphate of polyribonucleotides with hydrolysis of ATP toAMP and Ppi (Silber et al., 1972). The circularization reaction of theoligonucleotides by T4 RNA ligase provides some information on theoptimum physical distance between the donor and acceptor parts of an RNAmolecule, the optimum of which varies between a distance of 10-16nucleotides (Kaufmann et al., 1974, Sugino et al., 1977). The majorfeature that differs RNA ligase from DNA ligases is that RNA ligasealigns the free ends of the reacting donor and acceptor on its surface,whereas DNA ligase requires a base-paired template that fixes theoligonucleotide ends in close proximity (Engler and Richarson, 1982,Uhlenbeck and Gumport, 1982).

To create the optimal conditions for the RNA ligase, the claimed set ofdetector-molecules is designed with the option to bind the targetanalyte with a gap between them. Additionally, each of thedetector-molecules is designed with terminal nucleotides that should befree in a detector-analyte complex. Neither of these new structuralcomponents in the claimed detector-target complexes exists in standardnucleic acid probe-nucleic acid target complexes.

Preferably the RNA molecules do not align contiguously with each anotheron the target or on the analyte forming a gap between 20 and 200angstroms. Additionally, the RNA molecules align on the target or on theanalyte with free termini, that do not hybridize to the target moleculeor bind to the analyte.

Another embodiment of the present invention is the composition of thetwo detector-molecules that work in conjunction with each other whenthey are used in diagnostic protocols, kits or apparatuses. Each of thedetector-molecules consists of three functional parts. One of them is asegment of Q-beta replicase template, another is a segment of thesequence with high affinity to the target, and the third part is asegment of the sequences with the recognition site for a chosenendonucleatic compounds which, after the cleavage, acquires a captomer'sfunction. The first detector molecule is capable of binding to thetarget molecule and has the following formula, with at least threecomponents organized in a 5′->3′ direction as:

5′—A—B—C—3′.

As used above, A is a section of the first detector molecule having the5′ section of a Q-beta replicase template, which is not capable ofbinding to the target molecule and which is not capable of replication.The letter B denotes a section of approximately 20 to 50 nucleotides ofthe first detector-molecule which is the section capable of binding tothe target molecule. The letter C denotes a captomer, a section of thefirst detector composed of approximately 1 to 15 nucleotides, which isthe primary substrate for a ribozyme. The C captomer can and cannot becapable of binding to the target molecule.

The second detector-molecule is capable of binding to the targetmolecule and has the following formula with at least three componentsorganized in a 5′->3′ direction as:

5′—D—E—F—3′

As used above, the letter D denotes a captomer, a section of the seconddetector composed of approximately 1 to 15 nucleotides, which is anotherprimary substrate for a ribozyme. The D captomer can and cannot becapable of binding to the target molecule. The letter E denotes asection of the second detector-molecule having approximately 20 to 50nucleotides which section is capable of binding to the target moleculein the same “epitope” as sector B of the first detector molecule or toits own “epitope”. The F part of the second detector-molecule containsthe remaining 3′ section of a Q-beta replicase template. The A and Fsegments are not capable of binding to the target molecule and cannot beamplified separately by Q-beta replicase.

Preferably, sections C and D are the nucleotide sequences, termedcaptomers, that can serve as a donor, since section C naturallyterminates in the hydroxyl group required for ligation, and, section D,that can serve as an acceptor, with a terminal monophosphate grouprequired by the ligation reaction catalyzed by RNA ligase, whichincludes but is not limited to bacteriophage T4 DNA ligase. The ligationreactions are performed under standard conditions known to those skilledin the field of molecular biology.

Preferably, upon ligation of sections C and D the two detector-moleculesform a single molecule composed of five parts in the 5′-3′ order

5′—A—B—C—D—E—F—3′

as in the original recombinant RNA demonstrating high affinity to thetarget molecule.

Preferably, the newly formed molecule is a template amplifiable byQ-beta replicase and has a signal-generating moiety. Signal-generatingmoieties comprise, by way of example, radiolabeled nucleotides, enzymes,ligands, fluorescent or chemoluminescent agent, or sequences ofnucleotides capable of detection. A preferred signal-generating moietyis section A of the first detector molecule and section F of the seconddetector-molecule, which are two sections of the replicable nucleic acidtemplate. And, most preferably, sections A and F are selected from thenaturally-occurring group of replicable RNAs consisting of MDV-I RNA,Q-beta RNA, microvariant RNA, midivariant RNA, nanovariant RNA andmodifications thereof, or any artificially constructed RNA templatesthat permit the RNA to maintain replicable RNA. In the presence of theenzyme Q-beta replicase and suitable reaction conditions, preferredreplicable RNAs are produced and act as a signal that such replicableRNA is present initially as sections A and F in the twodetector-molecules and the reaction product in which it is incorporated.

A further embodiment of the present invention features a method ofdetermining the presence or absence of a target molecule. The methodcomprises the steps of providing first and second detector-molecules.The first detector-molecule is a non-naturally occurring, recombinantRNA molecule capable of binding to a target analyte and has a structure

5′—A—B—C—3′.

The segments A, B and C are as previously described. The second detectormolecule is a non-naturally occurring, recombinant RNA molecule capableof binding to the target analyte and has a structure:

5′—D—E—F—3′.

The sections D, E, F are previously described.

The method further comprises the step of imposing binding conditions ona sample potentially containing target molecules in the presence of thefirst and second detector-molecules. In the presence of the targetmolecule, the first and second detector molecules form a target firstand second detector-molecule complex. The method further comprises thestep of imposing RNA ligase reaction conditions on the sample to form athird RNA molecule in the presence of the target analyte. The third RNAmolecule has the formula:

5′—A—B—C—D—E—F—3′.

The sample is monitored for the presence of the third RNA molecule,whose presence or absence indicates the presence or absence of thetarget analyte molecule.

Preferably, sections B and E bind to the target analyte throughnon-nucleic acid pairing interactions. And, most preferred, B and E areligands demonstrating high affinity to separate epitopes of the sametarget analyte or they are parts of the same ligand that demonstrateshigh affinity to a single epitope of the target analyte.

Preferably, sections C and D are capable to serve as donor and acceptorcaptomers in the ligation reaction and can join covalently at therespective termini by the action of RNA ligase.

Preferably, at least one of the first or second detector-molecules has asignal-generating moiety. Preferably, the non-signal-generating firstand second detector-molecules are ligated and comprise a functioning,signal-generating template that can be amplified by Q-beta replicase.The analyte and the third RNA molecule are associated in the complex.All procedures of the binding of detector and analyte molecules andligation of the detector molecules are performed according to thestandard protocols known to the someone skilled in the art of molecularbiology.

A further embodiment of the present invention features the proceduresessential for the elimination of possible background. The methodpreferably comprises the further step of hybridization of a speciallydesigned primer with the complementary region of the C section prior tothe template's amplification by Q-beta replicase. For thishybridization, the mixture of detector-molecules annealed andnon-annealed to the analyte are mixed with the primer that hascomplementary nucleotide sequences and can bind to a segment of the CDregion of the ligated product. The mixture will be exposed to favorableconditions under which the primer will be reassociated with thecomplementary sequences of the CD region.

After primer reassociation conditions are imposed, the method preferablycomprises the further step of enzymatic degradation of the templatemolecules resulting from ligation of the first and seconddetector-molecules without binding to the analyte. Such molecules stillbear the signal-generating moiety and, therefore, could be the source ofbackground and false positive results. For this, the mixture will beexposed to AML reverse transcriptase as was described earlier.Similarly, the enzyme with its dual function will synthesize cDNAcomplementary to the CD, E and F, or A and B regions depending on theplus or minus nature of the ligated product, and will simultaneouslydegrade these segments of the RNA template, eliminating the 5′ portionof those template molecules not forming a complex with the targetanalyte.

After enzymatic degradation of the detector-molecules that are theligation products of the first and second detector-molecules in theabsence of the target analyte, the remaining templates are exposed toamplification by the Q-beta replicase enzyme.

A further embodiment of the present invention comprises a kit fordetermining the presence or absence of target molecules. The kit foridentifying specific analytes in a specimen is composed of one or morereagents comprising the first and second detector-molecules specific forthe desired or target analytes, RNA ligase, AML reverse transcriptase,Q-beta replicase, primer for the reverse transcriptase, and buffers,salt and reagent solutions necessary to perform the experimentsaccording to the designed protocol. The first detector molecules havethe formula:

A—B—C.

The second detector molecules have a formula:

D—E—F.

All segments of the detector molecules are previously described.

A universal kit for constructing the detector molecules for any analyteof interest ay be composed of one or more reagents comprising one of theRNA libraries, each species in which has a formula:

A—B—C—D—E—F.

The segments A, B, C, D, E and F are previously described. The kit alsoincludes RNA ligase, AML reverse transcriptase, Q-beta replicase, aribozyme that corresponds to the sequences in the CD region, primer forthe reverse transcriptase, and buffers, salt and reagent solutionsnecessary to perform the experiments according to the designed protocol.

It is possible to expect that some detector molecules will be ligatedand will form template molecules without annealing with the analytemolecule. Elimination of the background created by these “accidental”templates will be accomplished by enzymatic degradation and will beperformed prior to their amplification with Q-beta replicase. A similarapproach was used previously for differentiation of high and lowaffinity RNA recombinant molecules. After purifying the annealeddetector molecule-target analyte complex from the DNA and RNA fragments,non-annealed target analytes and detector molecules, the intact templatefor Q-beta replicase, protected by the target analyte, will be separatedfrom the target analyte. Amplifying the restored template with Q-betareplicase will indicate the presence of even a small number of targetanalyte molecules in a specimen.

In a modification of this method to produce two detector-molecules, therecombinant DNA molecules are transcribed into RNA before thereplication by Q-beta replicase. A RNA polymerase promoter is attachedto the nonreplicable portion of the ligated detector-molecules beforethe transcription. The attachment of the promoter and transcription ofrecombinant RNA are used under the standard conditions known to someoneskilled in the field of molecular biology.

EXAMPLE 1.

Using the above-cited specifics in the RNA ligase action, we performedan experiment in which the detector probes with captomers formed a“loose” ternary complex with a nucleic acid target and thus modeled acomplex formed by the detector-probes with a protein target. We alsoinvestigated the ability of T4 RNA ligase to use such a model complex asa substrate and to restore a template for Q-beta replicase by joiningspecially-designed captomers.

Preparation of the detector probes.

The fifty-base oligonucleotide sequence containing an Hha I- Pvu IIregion of the late promoter of adenovirus within map units 16.4 and 16.6(Ziff and Evans, 1978) was chosen as the model target in ourexperiments. It was synthesized on a DNA Synthesizer, together with twopairs of oligonucleotides—oligos #1 and #2 and oligos #3 and #4. Thefifty base oligonucleotiding (Seq ID No 5) is described below with theposition of Oligos, #1-4.

Seq. ID No. 5   1       10       20          30        40         505′-CGCGTTCGTCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGGGCC-3′  |<----------------->|    |<---------------------->|      Oligo #3 and#4                 Oligo #1 and #2

The first pair of oligos complement each other and represent thecounterparts of the adenovirus target region from the nucleotide C²⁵ tothe end of the sequence. Oligos #1 (Seq ID No. 6) and Oligo #2 (Seq IDNo 7) are described below:

Seq. ID NOS 6 and 7 Oligo #1    Xhol     10        20        30     375′-TCGAGGCCCTGGCCCTCGCAGACAGCGATGAGCTCCC-3′ Oligo #2   3′-CCGGGACCGGGAGCGTCTGTCGCTACTCGAGGG-5′       33 30       20        10 Sacl Smal                                (Sstl)

Both oligos have additional sequences representing the complete site forSac I and a half for Sma I restriction enzymes, and the oligo #1additionally has a sequence of the Xho I restriction enzyme.

Oligos #3 and #4 represent the other half of the adenovirus targetmolecule and span from the beginning of the target sequence to the T²¹base. Oligos #3 (Seq. ID No. 8) and Oligo #4 (Seq. ID No. 9) aredescribed below:

Seq. ID Nos 8 and 9         PpuM-1       10        20 22 Oligo#3  5′-AAGAGAGTGAGGACGAACGCGC-3′ Oligo#4  3′-TTCTCTCACTCCTGCTTGCGCGAGCT-5′             26      20      10    Xhol

Both oligos #3 and #4 have half of the recognition site for the PpuM-Irestriction site and oligo #4 has a sequence of Xho I restrictionenzyme, similar to oligo #1. These two pairs of oligos were alsoannealed and were used for cloning. They are referred to as the “PXfragment”. These two pairs of oligos formed two dsDNA fragments,referred to as the “PX fragment” and “XS fragment”. They were used forcloning in the recombinant plasmid pT7 MDV-XhoI.

Turning now to FIG. 1, which depicts plasmid pT7 MDV-XhoI, this plasmidwas used for cloning of the synthesized PX and XS dsDNAs and for thetranscription of the PX and XS recombinant RNA molecules that served asdetector molecules for the adenovirus sequences. This plasmid is avariant of the parent plasmid, pT7 MDV, in which the XhoI linkersequence is not present. For further details of the construction of thevector, see Axelrod et al., 1991. A sequence described in FIG. 1 isdesignated Seq. ID No. 22.

The pT7MDV-Xho I plasmid DNA was digested either with Ppu MI and Xho Ior with Sma I and Xho I restriction enzymes and purified from theexcised 65 bp or 166 bp fragments of the cloned MDV cDNA insert. Theremaining parts of the pT7MDV-XhoI plasmid after Ppu MI and Xho Idigestion (pT7 MDV-1) contained the T7 promoter and 168 bp 3′ end of theMDV cDNA. The remaining part of the pT7MDV-Xho I plasmid digested withXho I and Sma I enzymes (pT7 MDV-2) contained T7 promoter and the 62 bp5′ end of the MDV cDNA. The PX fragment was ligated with the pT7 MDV-1and the XS fragment was ligated with pT7 MDV-2, forming pPX and pXSrecombinant plasmids. The presence of the two different cloning sites inthe digested vector ensures only one possible orientation of the insertsin both the pPX and pXS plasmids. Each of these plasmids contains aT7RNA promoter, an insert homologous to part of the target adenovirussequence, and a segment of the MDV cDNA.

A control recombinant plasmid with target adenovirus DNA inserts wasconstructed as well. For this purpose, we modified the originaladenovirus sequence so that it contained Xho I sites at both endssimilarly to PX and XS fragments. The fragment was then inserted at theXho I site of pT7 MDV- Xho I, creating plasmid p325. This plasmid wasused either to produce the DNA target sequences or to synthesize therecombinant template MDV RNA with an insert of the adenovirus sequences.

Cold or ³²P-labeled RNA was transcribed from plasmids using T7RNApromoter after the Sma I digestion of pPX or p325 and Sst I or Sma I ofpXS digestion according to the standard protocols (Sambrook et al.,1989)

The 193-base RNA transcript from the pPX plasmid is composed of (readingin the 5′♯3′ direction) the first three G residues of transcriptioninitiation, 22 residues transcribed from the PX fragment and 168residues transcribed from the 3′ end of the MDV cDNA. This sequence(Seq. ID No. 10) is described below:

5′-GGG-AAGAGAGUGAGGACGAACGCGC-3′ MDV-1 RNA (168 bases).

The RNA transcript from the pXS plasmid is only 92 bases, shorter thanthe pT7MDV-1 transcript, and it starts with the 62 residues of the 5′end MDV cDNA and is followed by the 30 residues transcribed from the XSfragment. This sequence (Seq. ID No. 11) is described below:

5′-MDV-1 RNA (62 bases)-GGCCCUGGCCCUCGCAGACAGCGAUGAGCU-3′.

An additional three C nucleotides were generated on 3′ end of the XSrecombinant RNA after Sma I digestion of the same plasmid.

These two recombinant RNAs represent a set of the PX and XS detectormolecules. Each of these molecules has three functional parts:amplification, recognition and ligation.

Annealing experiments

PX detector was ³²P end-labeled to monitor the results of the annealingexperiments. For this we used bacterial alkaline phosphatase (BAP) andbacteriophage T4 polynucleotide kinase. Both reactions,dephosphorylation and end-labeling, were performed according to standardprotocols (Sambrook et al., 1989). Incorporation of the radioactivelabel was measured in aliquots of the reaction and the percentages ofincorporation were calculated.

The PX and SX detector molecules were hybridized with the targetadenovirus oligonucleotide sequences. For this the three components weremixed in equal molarity proportion in a range of 2-3 pmole per reaction.

The hybridization produces several radioactive bands, representing theproducts of association between the target adenovirus DNA and the PX andXS probes.

The annealing reaction products between the PX and XS detector-moleculesand adenovirus target sequences were characterized. PX recombinant RNAwas ³²P end-labeled using standard methods. Annealing was achieved byboiling the reaction mix two minutes and then incubating it at 65° C.for two hours. The annealing reaction was carried out in a solutioncontaining 50 mM TRIS pH 7.8, 5 mM MgCl₂, 0.5 mM ATP and 1 mM EDTA, 10%non-denaturing PAGE at 500 volts for eight hours.

The most plausible explanation of the results is that a top band resultsfrom the annealing of the target DNA molecule with both RNAs. Suchcomplex is composed of from 50 bp double stranded heteroduplex oftarget/probes segment and 168 bases 3′-end and 62 bases 5′-end of MDV-I.A lowest band is 193 nucleotides of non-hybridized PX RNA and one of themiddle bands, with a size of 243 bases, is a complex between PX RNA andadenovirus DNA molecules. The origin of a second band of similar size isunknown.

The efficiency of hybridization was calculated as a percentage of theradioactivity of the top band from the total radioactivity applied onthe gel. Usually more than 50% of the total number of PX RNA moleculesparticipate in hybridization with the adenovirus target molecule byitself or in compound with XS. The yield of the ternary complex formedby two RNA detector molecules and the target molecules was, usually,close to 30-40%.

FIG. 2 illustrates a possible hybridization configuration between thetarget adenovirus sequence and the two RNA transcripts. The SX RNAdetector, in this case, was generated by Sst I digestion of the pSXplasmid. The ligated first and second RNA molecules are identified inSeq. ID No. 12.

SEQ ID No. 12

5′-GGCCCUGGCC CUCGCAGACA GCGAUGAGCU GGGAAGAGAG UGAGGACGAA CGCG-3′

There is complete complementarity along the PX RNA detector and thefirst 23 bases of the target molecule, but not the last, the 24thG-residue of the transcript and the G-base of the adenovirus targetmolecule. The first four bases of the XS detector (UCGA) do not havehomologous nucleotides on the target DNA molecule, although the rest ofthe transcript is complementary to the target molecule. Thus, thehybridized RNA transcripts do not juxtapose to the target end-to-endfashion, but rather leave a ˜20 Angstrom gap between the terminalhybridized nucleotides.

The G and UCGA nucleotides of the PX and XS detector probes are thecaptomers. They do not hybridize to the target and comprise structuressimilar to the donor/acceptor complex, which is necessary in order forRNA ligase to form a phosphodiester bond (Uhlenbeck, 1983). The Gcaptomer is a donor with a 5′-phosphate terminus and the UCGA captomeris an acceptor with a 3′-hydroxyl terminal group on the U residue.

Ligation experiments

The recombinant plasmid pXS was constructed in a such way that it couldbe linearized either with SmaI or SstI for RNA transcription. The two XSRNA detector molecules are different in the total lengths andcomposition of their captomers. The XS detector generated with Sst Idigestion had a captomer of four UCGA-base-long bases, whereas thecaptomer generated after the Sma I digestion was longer for the threeCs.

Ligation reactions were carried out on 4 ul aliquots taken directly fromthe annealing reactions in the presence of the 10 nM mercaptoethanol,and 40 Units of T4 RNA ligase at 25° C. after confirmation by gelelectrophoresis that hybridization was successful. The duration of thereaction varied from 2 hours to overnight. The bands representingligation product composed of PX-SX ligated detector molecules wereexcited and their radioactivity was measured and compared with the totalradioactivity of the aliquot from the annealing reaction used for theligation. The total volume of each ligation reaction was 10 ul, with thefinal concentrations 10 mM, 5 mM MgCl₂ and 2 Units of T4 RNA ligase inthe presence of 20% PEG. The reaction was performed at 25° C. and endedby adding 1 ul of 100 mM EDTA, 7M Urea denatured 10% PAGE at 500 voltsfor eight hours of electrophoresis.

Additionally, the duration of the reaction apparently does not affectthe rate of ligation when the long captomers were used. The yield of theligated product was 20.0% after two hours of reaction and 18.7% afterovernight. The longer reaction time, however, might have a certaindisadvantage when the short captomers are used. The overnight reactionyielded 18.4% compared with 33.2% after two hours of reaction. Thereduction in the percentages of ligated products after a prolongedreaction time apparently indicate that the ligation products composed ofPX and XS RNA transcripts are not stable and dissociate over time. Theresults of the ligation experiment demonstrates that the length of theXS captomer seemingly does not effect the ligation rate of the RNAtranscripts, although the highest ligation rate was observed when theacceptor-captomer was composed of the seven-AGCUCCC- residues.³²P-labeled recombinant MDV-I RNA, with the adenovirus inserttranscribed from the p325 plasmid, served as a reference marker.

TABLE 1 Effect of the captomer's length on the yield of the ligationproducts between PX and XS RNA transcripts. 4 ul aliquots of theannealing reactions (1-6*) with two RNA transcripts and adenovirustarget DNA were used for the subsequent ligation reaction. 4 ul aliquotsof the annealing reaction (7**) without the target DNA were used as thenegative control. Length Total of captomer Duration counts (cpm) Theirproportion used of reaction of the band (%) from the Test # in the at25° C. in the gel reaction counts loaded 1. Short¹ 2 hours 3428 20.0% 2.Short o/n 3140 18.7% 3. Long² 2 hours 5576 33.2% 4. Long 2 hours 16579.9% 5. Long o/n 3085 18.4% 6. Long o/n 1385 8.2% 7. Long 2 hours 980.01% *16.800 cpm were loaded into each test lane **76.700 cpm wereloaded into a control lane

The short (UCGA) captomer was generated by the pXS plasmid DNA digestionby the Sst I restriction enzyme, and the long (AGCUCCC) captomerresulted from the digestion of the pXS plasmid DNA by the Sma Irestriction enzyme.

The ligation product composed of the ligated PX and XS RNA detectormolecules was purified by gel electrophoresis. The purified product wasused as a template for Q-beta replicase.

Amplification of the ligation products by Q-beta replicase

Q-beta replicase reactions were carried out on a volume of 20 ul at 37°C. during 25-30 minutes in 50-ul reactions containing 88 mM Tris-HCL (pH7.5), 12 mM MgCl₂, 0.2 mM of each ribonucleoside triphosphate, 25 uCi of[alpha-³²P]GTP, 90 pm/ml of Q-beta replicase, and 11.2 pm/ml of templateRNA. From this mixture, 7 to 15 ul was applied directly onto adenaturing polyacrylamide gel containing 7M Urea for electrophoreticanalysis. Additionally, adsorbed radioactivity was determined by liquidscintillation.

The Q-beta replicase experiment demonstrates that there are no templatesfor Q-beta enzyme in the aliquots representing the tube in which theligation was performed without the adenovirus target, which indicatethat target analyte was necessary to unite two detector probes. Aligation reaction was performed on aliquots of the annealing reaction.The 5 ul aliquots from each reaction were analyzed on a non-denaturinggel.

The obtained data suggest that amplification of the template by Q-betareplicase occurred only when ligation of the PX and XS RNA detectormolecules took place in the presence of the target.

The present invention features a “flexible” detector-target ternarycomplex and captomers sections. These features permit use of RNA ligaseto restore the ability of recombinant MDV-1 RNA to be a template forQ-beta replicase.

EXAMPLE 2.

A Q-beta replicase-based system of detector-molecules can be constructedand used to identify a protein target in a clinical specimen. As anexample we use the high affinity RNA ligand (RRE-aptamer) for Revprotein. The central role of Rev protein in primary and reactivatedhuman immunodeficiency virus type 1 (HIV-1) infections is well studied.The interaction of Rev protein and the cis-acting Rev Responsive Element(RRE) is required for cytoplasmic accumulation of HIV structuralproteins encoding viral mRNAs (Malim et al., 1989. The Rev protein hasbeen isolated and purified and a series of high affinity aptamers havebeen identification.

The structure of RRE and the mechanism of interaction between RREaptamers and Rev protein is well studied. (Karn et al., 1991). The RREand its aptamers consist of three functional parts. One “bubble” (thecore region), and two double-stranded stems that flank the core region.Experiments with nucleotide substitution showed that the annealingabilities of the whole aptamer depends on the existence of a coreregion, its nucleotide composition and tertiary configuration. Thenucleotide composition of the flanking stems is not crucial as long asthe stems ensure the existence of the core region (Giver et al., 1993 a,b ). SELEX-derived oligonucleotides with K_(d) are about 10 times lowerthan those of wild type RRE (Giver et al. ibid.). Therefore, RREaptamers can successfully compete with native RRE for the Rev protein.The Rev protein system and high affinity RNA ligands selected in vitroare used by many research groups as targets for designing an anti-HIVstructure-based drug (Klug and Famulok, 1994).

Turning to FIG. 3, RBE-2, a high affinity aptamer selected throughSELEX, preserves the structure of the RRE core element, which issupported by two double-stranded stems, and demonstrates high affinityto Rev protein (Bartel et al.,1991). The nucleotides in the core regionU⁴⁵♯G⁵³ and C⁶⁵♯A⁷⁵, which are involved in the binding motif for Revrecognition, are shown in italics. A major groove formed in the coreregion by the two G bases, is underlined and bold in the diagram. Thesequence is identified as Seq. ID No. 13.

SEQ. ID No. 13

5′-GGUGGGCGCA GCGUCAAUGA CGCUGACGGU ACACC-3′

These two nucleotides open or pull apart the otherwise stringed stem ofcomplementary nucleotides. Such widening is necessary for precisespatial positioning of the Rev peptide's “epitope” in the major groove.Finally, hydrogen bonds form between the Rev protein and RNA, securing atight and specific binding into the complex (Iwai et al., 1992).

To construct recombinant RNA molecules containing the aptamer sequencesfor Rev protein and sequences for the template for Q-beta replicase, thedsDNA representing the whole aptamer with terminal XhoI sites, and thetwo dsDNAs representing the halves of the aptamer, are synthesized.These, too, will have appropriate cloning restriction site terminals.The three oligonucleotides are cloned in a pT7 MDV-Xho I plasmid (FIG.1), using the same strategy and procedures that were performed in modelexperiments with detector probes specific for the adenovirus sequences.A dsDNA (Seq. ID Nos. 14 and 19) corresponding to a first aptamer isdepicted below:

Seq. ID Nos. 14 and 19   Xhol Rev protein aptamer5′--TCGA--GGTGGGCGCAGCGTCAATGACGCTGACGGTACACC--3′ dsDNA     3′--CCACCCGCGTCGCAGTTACTGCGACTGCCATGTGG-AGCT-5′                                                                   Xhol

A second dsDNA (Seq. ID Nos. 15 and 20) corresponding to a secondaptomer is described below:

Seq. ID Nos. 15 and 20   Xhol dsDNA of first5′--TCGA--GGTGGGCGCAGCGTCAA--AGCT-3′ detector molecule3′--CCACCCGCGTCGCAGTT--TCGA-5′                         Sstl

A third dsDNA (Seq. ID No. 16 and 21) corresponding to a third aptomeris described below:

Seq. ID Nos. 16 and 21   PpuMl dsDNA of second5′--AA--TGACGCTGACGGTACACC--3′ detector molecule3′--TT--ACTGCGACTGCCATGTGG-AGCT-5′                            Xhol

RNA detector probe transcripts, composed of the RBE-2 and Q-betareplicase template sequences, will be synthesized for each pair ofoligos, similar to those of the PX and XS for adenovirus, using the T7RNA promoter and T7RNA polymerase. The composition of the recombinantRNA transcripts representing the two recombinant RNA detector molecules(Seq. ID No. 17 and 18) are described below:

Seq. ID Nos. 17 and 18 5′--62ntMDV--GGUGGGCGCAGCGUCAA--AGCU--3′5′--GGG--AA--UGACGCUGACGGUACACC--168ntMDV--3′

The FIG. 3a demonstrates the possible configuration of the ternary Revprotein-detector probes complex. The detector molecules would be ligatedwith T4 RNA ligase and will form a single molecule as shown in FIG. 3band described in the Sequence Listing as Seq. ID No. 23.

The newly constructed aptamer is different from the original by theelongation of the stem between the core region by two base pairs and theloop by five bases, and by the presence of MDV RNA sequences. Itscomposition is 5′-62ntMDV-RBE-2-168ntMDV-3′. It will be released in alow-salt buffer and MDV reporter templates can be amplified with Q-betareplicase without any additional processing. The number of reportingligated detector molecules presented in the eluted sample will beproportional to the number of Rev protein molecules in the model samplesor clinical specimens, and can be estimated by adding the sample to astandard reaction mixture at 37° C. containing Q-beta replicase andmeasuring the time required to produce a signal with an intercalatingfluorescent dye. Appropriate negative and positive standards will betested simultaneously with the specimens to establish sensitivity andspecificity levels of the assay and to find a correlation between theassay results and the actual HIV titer in the clinical specimens. On theserially diluted Rev protein samples, we will establish a relationshipbetween response time and the number of target molecules in the sample.The response time is universally proportional to the log of the numberof template molecules present in the sample (Lomeli et al., 1989). Thefluorescence of the amplified detector molecules will be photographedover an ultraviolet light box or measured in a fluorometer. The resultsof these experiments will enable us to identify the conditions necessaryto form the preferred ternary complex of Rev protein and recombinant RNAdetector probes with minimal background for the whole assay and,therefore, with high specificity for the whole assay.

Thus, while preferred embodiments have been illustrated and described,it is understood that the present invention is capable of variation andmodification and therefore, should not be limited to the precise detailsset forth, but should include such changes and alterations that fallwithin the purview of the following claims.

REFERENCES U.S. PATENT DOCUMENTS

Cech T. R., Murphy F. L., Zaug A. J., Grosshans C., 1992. RNA ribozymerestriction endonucleases and methods. U.S. Pat. No. 5,116,742

Gold L. and C. Tuerk. 1995. Nucleic Acid Ligands. U.S. Pat. No.5,475,096

Gold L. and S. Rinquist. 1996. Systematic Evolution of Ligands byExponential Enrichment: Solution SELEX. U.S. Pat. No. 5,567,588.

Hazeloff J. P., Gerlach W. L., Jennings P. A., Cameron F. H. 1993.Ribozymes. U.S. Pat. No. 5,254,678.

Roberson H. D., and Goldberg A. R., 1993. Ribozyme Composition andMethods for Use U.S. Pat. No. 5,225,337

OTHER PUBLICATIONS

Axelrod V. A., Brown E., Priano C. and Mills D. R. 1991. Virology, 184,595-608

Bartel D. P., Zapp M. L., Green M. R. and J. Szostak. 1991. Cell 67,529-536.

Ellington A. D. and Szostak J. W. 1990. Nature, 346, 818-822.

Giver L., Bartel D. P., Zapp M. L., Green M. and Ellington A. D. 1993b.Gene 137,19-24

Gold L., Polisky B., Uhlenbeck O., and Yarus M. 1995. Ann. Rev. Biochem.64, 763-797.

Iwai S., Pritchard C., Mann D. A., Karn J. and Gait M. J. 1992. NucleicAcid Res. 20, 6465-6472.

Joyce, G. F. 1989. Gene, 82, 83-87.

Karn J., Dingwall C., Finch J. T., Heaphy S. and Gait M. J. 1991.Biochimie 73,9-16.

Kaufmann G., Klein T. and Littauer U. Z. 1974. FEBS Lett. 46, 271-275.

Klug S. J. and Famulok M. 1994. Mol. Biol. Rep., 20, 97-107

Leis J., Silber R., Malathi V. G. and Hurwitz J. 1972. AAdvances in theBiosciences@ (G. Raspe, ed) Pergamon, New York. vol. VIII, 117

Malim M. H., Hauber J, Le S. Y. Maizel J. V. and Cullen B. R. 1989.Nature 338, 254-257.

Meselson M. and Yuang R. 1968. Nature, 217, 1110-1114

Nakamura R. M. 1993. College of American Pathologists Conference XXIV onMolecular Pathology: Introduction. Ach. Path. Lab. Med., 117, 445-492

Sambrook J., Fritsch E. F. and T. Maniatis. 1989. Molecular Cloning.Cold Spring Harbor Laboratory Press.

Silber R. Malathi V. G. and Hurwitz J. 1972. Proc. Natl. Acad. Sci. USA69, 3009-3013

Southern E, 1975. J. Mol. Biol., 98, 503-517.

Sugino A., Goodman H. M., Heyneker H. L., Shine J., Boyer H. M. andCozzarelli N. R. 1977. J. Biol.Chem. 252, 3987-3987

Tuerk C. and Gold L. 1990. Science. 249. 505-510.

Uhlenbeck O. C., and Gumport R. D. 1982. The enzymes. Academic Press,Inc. vol XV. 31-58.

Uhlenbeck O. C. 1983. TIBS. March, 94-96.

Verma I. M. 1991. The Enzymes, The Academic Press, vol XIV, 87.

Ziff E. B. and Evans R. M. 1978. Cell 15, 1463-1475.

23 1 69 RNA Artificial Sequence Description of Artificial SequenceQ-beta phage partial template 1 ggggaccccc ccggaagggg gggacgaggugcgggcaccu cguacgggag uucgaccgug 60 acgcucuag 69 2 166 RNA ArtificialSequence Description of Artificial Sequence Partial template for Q-betaphage. 2 agaucuagag cacgggcuag cgcuuucgcg cucucccagg ugacgccucgugaagaggcg 60 cgaccuucgu gcguuucggu gacgcacgag aaccgccacg cugcuucgcagcguggcucc 120 uucgcgcagc ccgcugcgcg aggugacccc ccgaaggggg guuccc 166 337 RNA Artificial Sequence Description of Artificial Sequence Partialtemplate for Q-beta phage. 3 ggggaaaucc uguuaccagg auaacggggu uuccuca 374 54 RNA Artificial Sequence Description of Artificial Sequence Partialtemplate for Q-beta phage. 4 ccucucuacu cgaaaguuag agaggacaca cccggaucuagccgggucaa ccca 54 5 50 DNA Artificial Sequence Description ofArtificial Sequence Partial sequence derived from adenovirus. 5cgcgttcgtc ctcactctct tccgcatcgc tgtctgcgag ggccagggcc 50 6 37 DNAArtificial Sequence Description of Artificial Sequence Probe sequence tosection of adenovirus nucleic acid. 6 tcgaggccct ggccctcgca gacagcgatgagctccc 37 7 33 DNA Artificial Sequence Description of ArtificialSequence Probe sequence to adenovirus nucleic acid 7 gggagctcatcgctgtctgc gagggccagg gcc 33 8 22 DNA Artificial Sequence Description ofArtificial Sequence Probe sequence to adenovirus nucleic acid. 8aagagagtga ggacgaacgc gc 22 9 26 DNA Artificial Sequence Description ofArtificial Sequence Probe sequence to adenovirus nucleic acid. 9tcgagcgcgt tcgtcctcac tctctt 26 10 25 RNA Artificial SequenceDescription of Artificial Sequence Binding sequence to adenovirusnucleic acid. 10 gggaagagag ugaggacgaa cgcgc 25 11 30 RNA ArtificialSequence Description of Artificial Sequence Binding sequence toadenovirus nucleic acid. 11 ggcccuggcc cucgcagaca gcgaugagcu 30 12 54RNA Artificial Sequence Description of Artificial Sequence Ligatedbinding sequences. 12 ggcccuggcc cucgcagaca gcgaugagcu gggaagagagugaggacgaa cgcg 54 13 35 RNA Artificial Sequence Description ofArtificial Sequence Aptamer nucleic acid to Rev protein. 13 ggugggcgcagcgucaauga cgcugacggu acacc 35 14 39 DNA Artificial Sequence Descriptionof Artificial Sequence Rev protein aptamer nucleic acid. 14 tcgaggtgggcgcagcgtca atgacgctga cggtacacc 39 15 25 DNA Artificial SequenceDescription of Artificial Sequence Rev protein aptamer nucleic acid. 15tcgaggtggg cgcagcgtca aagct 25 16 20 DNA Artificial Sequence Descriptionof Artificial Sequence Rev protein aptamer nucleic acid. 16 aatgacgctgacggtacacc 20 17 21 RNA Artificial Sequence Description of ArtificialSequence Rev protein detector molecule nucleic acid. 17 ggugggcgcagcgucaaagc u 21 18 23 RNA Artificial Sequence Description of ArtificialSequence Rev protein aptamer detector molecule nucleic acid. 18gggaaugacg cugacgguac acc 23 19 39 DNA Artificial Sequence Descriptionof Artificial Sequence Rev protein aptamer nucleic acid. 19 tcgaggtgtaccgtcagcgt cattgacgct gcgcccacc 39 20 21 DNA Artificial SequenceDescription of Artificial Sequence Rev protein aptamer nucleic acid. 20agctttgacg ctgcgcccac c 21 21 24 DNA Artificial Sequence Description ofArtificial Sequence Rev protein aptamer nucleic acid. 21 tcgaggtgtaccgtcagcgt catt 24 22 73 DNA Artificial Sequence Description ofArtificial Sequence Partial sequence derived from adenovirus. 22agcttgggct gcaggtctaa tacgactcac tataggggac cccccgagtc ctcgaggagt 60caccccggga att 73 23 21 RNA Artificial Sequence Description ofArtificial Sequence Ligated detector molecules. 23 ggugggcgca gcgucaaagcu 21

What is claimed is:
 1. A method of determining the presence or absenceof a target molecule, said method comprising the following steps: a.)providing a first RNA molecule and a second RNA molecule, said first RNAmolecule capable of binding to a target molecule and having thefollowing formula: 5′—A—B—C—3′; wherein A is a section of said RNAmolecule having 10 to 100,000 nucleotides which section is capable ofbeing received by an RNA replicase and with another RNA sequence, F,being replicated and the letter “B” denotes a section of the RNAmolecule having approximately 10 to 50,000 nucleotides which section iscapable of binding to the target molecule and the letter “C” denotes asection of the RNA molecule having approximately 1 to 10,000 nucleotideswhich section is capable of being ligated to another RNA sequence, “D”;said second RNA molecule capable of binding to said target molecule andhaving the following formula: 5′—D—E—F—3′; wherein D is a section of theRNA molecule having approximately 1 to 10,000 nucleotides which iscapable being ligated to another RNA sequence, “C”, and the letter “E”denotes a section of the RNA molecule having approximately 10 to 50,000nucleotides which section is capable of binding to the target molecule,and the letter “F” denotes a section of the RNA molecule capable ofbeing received by an RNA replicase and with another sequence, “A”, beingreplicated; said first and the second RNA molecules are capable offorming a third RNA molecule having the following formula:5′—A—B—C—D—E—F—3′; said third RNA molecule formed by ligating the C andD sections, as the E and the B sections are bound to target, said thirdRNA molecule capable of being received by an RNA replicase and beingreplicated by such enzyme as a indication of the presence or absence ofthe target molecule; b.) combining a sample potentially containing thetarget molecule with said first and said second RNA molecules andimposing conditions which allow said first and said second RNA moleculesand said target molecule to form a first second RNA molecule targetcomplex; c.) imposing RNA ligase conditions on said sample to form saidthird RNA molecule in the presence of the target molecule; d.) imposingamplification conditions on said sample to form an amplification productin the presence of target; and e.) monitoring the sample for thepresence or absence of the third RNA molecule as indicative of thepresence or absence of the target molecule.
 2. The method of claim 1further comprising the step of removing first and second RNA moleculeswhich do not form a complex.
 3. The method of claim 2 wherein said firstand second RNA molecules which do not form a complex are enzymaticallydestroyed.
 4. The method of claim 3 wherein said first and second RNAmolecules are destroyed by combining said sample and said first andsecond RNA molecule with the enzyme reverse transcriptase and imposingreverse transcriptase reaction conditions on said sample.
 5. The methodof claim 1 wherein said amplification conditions comprise combining saidsample potentially containing said third RNA molecule with the enzymeQ-beta replicase.
 6. The method of claim 1 wherein said section denotedby the letters “B” and “E” are aptamers or partial aptamers.
 7. Themethod of claim 1 wherein the section denoted by the letters “C” and “D”represent together define a sequence for cleavage by a ribozyme oranother compound which has an endonucleolytic activity against asingle-stranded nucleic acid.
 8. The method of claim 1 wherein thesections denoted by the letters “A” and “F” represent sequences selectedfrom the group consisting of MDV-1 RNA, Q-beta RNA, microvariant RNA,midivariant RNA, nanovariant RNA or modifications thereof which permitthe RNA to maintain its reproducibility.